A method that allows partial denaturation of protein ligands in Bi– and Zn–protein complexes, leaving the metal coordination centre intact, was developed. It was based on the reduction of the S–S bridges with tris(2-carboxyl)phosphine followed by derivatization with iodoacetamide. Consequently conditions that allow the separation of Bi– and Zn–protein complexes using SDS electrophoresis were found. The separation efficiency was much higher than that in non-denaturating blue native electrophoresis. The method allowed the detection of seven Bi-binding protein candidates in H. pylori treated with bismuth subcitrate, some of which—fructose-bisphosphate aldolase (33.6 kDa), urease alpha subunit (26.4 kDa), and the 16.8 kDa proteins: 30S ribosomal protein S6 and neutrophil activating protein (NapA)—were bio-induced during the treatment. The method also allowed the monitoring of the changes in the Zn-proteome during treatment of H. pylori with the Bi-drug, which was found to increase the concentration of the Zn-binding proteins with particularly strong expression of the urease, S-adenosylmethionine synthetase and the above 16.8 kDa proteins.
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